Monoclonal antibodies against leukocyte adhesion receptor beta-chain, methods of producing these antibodies and use therefore

ABSTRACT

Monoclonal antibodies and method for ameliorating an immune response disorder. The monoclonal antibodies are specific for an epitope present on the leukocyte adhesion receptor β-chain.

FIELD OF THE INVENTION

[0001] This invention relates to monoclonal antibodies specific for anepitope on the leukocyte adhesion receptor β-chain which can be used tosuppress intercellular leukocyte adhesion.

DESCRIPTION OF THE BACKGROUND ART

[0002] Human immunodeficiency virus (HIV) is the etiologic agent ofacquired immunodeficiency syndrome (AIDS), a fatal disease characterizedby profound immunosuppression, opportunistic infections, andneuropathies. Although only a small fraction of circulating lymphocytesare infected with the virus, there is a marked loss of T cells bearingthe virus receptor CD4. The depletion of CD4⁺-T cells appears tocontribute significantly to the immunosuppression associated with AIDS.Syncytium formation resulting from HIV-induced cell fusion has beenshown to be the primary cytopathic effect of the virus in vitro and hasbeen postulated to account for the loss of CD4⁺-T cells in vivo. CD4through its interaction with the HIV envelope glycoprotein gp120 playsan important role in syncytium formation.

[0003] Although the CD4 receptor appears to play a significant role inthe etiology of AIDS, several observations suggest that molecules on thesurface of uninfected cells other than CD4 are also involved inHIV-induced cell fusion. First, fusion of HIV-infected cells touninfected cells does not correlate with CD4-density on the surface ofthe uninfected cells. In addition, whereas transfection of non-lymphoidhuman cells with CD4 receptors renders such cells capable of fusion toHIV-infected cells, this is not true for CD4-transfected mouse cells.Finally, there is a disparity in the capacity of sera from AIDS patientsto block binding of HIV particles to CD4⁺-cells and the capacity of thesame sera to block fusion of HIV-infected cells to CD4⁺-uninfectedcells.

[0004] CD4 interacts directly with class II major histocompatibilitycomplex (MHC) molecules in class II MHC-restricted T helper cellresponses. The involvement of the leukocyte adhesion receptor (LAR)LFA-1, in such responses has been demonstrated using anti-LFA-1monoclonal antibodies (mAb). Structural similarities between gp120 andclass II MHC suggested that the binding of gp120 to CD4 may mimic theinteraction between class II MHC molecules and CD4. By analogy, the roleof LAR in HIV-mediated cell fusion was examined. In the presentinvention, a mAb against LFA-1 completely inhibits HIV-mediated fusionof uninfected T cell blasts to HIV infected cells. This result indicatesthat LFA-1 is involved in HIV-induced syncytium formation, a majorcytopathic mechanism of the virus.

[0005] The LFA-1 molecule, which is expressed on T and B lymphocytes aswell as on macrophages, thymocytes, granulocytes, and a subpopulation ofbone marrow cells, is composed of two non-covalently associatedpolypeptides of 175,000 Kd (α; CD11a) and Kd (β; CD18). The β-chain ofLFA-1 is also common to two other leukocyte antigens: Mac-1 (α-chain165,000 Kd; CD11b); the type-three complement receptor; and LeuM5(α-chain 150,000 Kd; CD11c), a molecule possibly associated withtype-four complement receptor activity. Although the three α-subunitsdiffer in size, there is evidence suggesting that all three subunits areencoded by a single gene or duplicated genes. cDNA encoding the humanβ-chain has been cloned and found to be 50% identical in primarystructure to the β-chain of integrin, a chick fibroblast fibronectinreceptor. These studies and others have shown that molecules of theLFA-1 glycoprotein family are members of the largerarginine-glycine-aspartate (RGD) adhesion family known as integrins.

[0006] At present, methods of limited effectiveness exist for thetreatment of AIDS or other disorders in which the intercellularinteraction of lymphocytes helps to mediate the pathologic state. Thosedrugs which are administered generally have severe contraindicationsassociated with their use. Consequently, a considerable need exists fora therapeutic agent which can inhibit lymphocytic intercellularinteraction in AIDS and other immune response mediated disorders.

SUMMARY OF THE INVENTION

[0007] One way to ameliorate immune response mediated disorders would beto suppress intercellular leukocyte adhesion using a monoclonal antibodywhich binds to a leukocyte adhesion receptor. In so doing, intercellularleukocyte binding is suppressed thereby decreasing the likelihood ofcell-to-cell transmition of infectious agents and immune responseactivation.

[0008] In order to provide a means to ameliorate immune responsemediated disorders, the inventor has developed monoclonal antibodieswhich bind to an epitope on the leukocyte adhesion receptor andsuppresses the ability of leukocytes to adhere to each other. Thesemonoclonal antibodies, if desired, can be therapeutically ordiagnostically labelled.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1. Inhibition of syncytium formation by mAb.

[0010]FIG. 2. Dose dependent inhibition of syncytium formation by H52IgG.

[0011]FIG. 3. H52 blocks syncytia formation at the level of PHA-blastsnot 8E5 cells.

[0012]FIG. 4. Inhibition of gp120 binding to CEM cells by mAb.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to monoclonal antibodies withspecificity for leukocyte adhesion receptor β-chain. These monoclonalantibodies are highly useful for both the in vitro and in vivoimmunological detection of antigens having these β-chains and forimmunotherapy of cells bearing these receptors having these β-chains.

[0014] In a preferred embodiment of the invention a monoclonal antibody(H52) is disclosed which binds to an epitope on the leukocyte adhesionreceptor β-chain. This specificity enables H52, and like monoclonalantibodies with the specificity of H52, to be used to suppressintercellular adhesion. As a consequence, H52 is useful in amelioratingimmune response mediated disorders such as AIDS, autoimmune disease, andgraft, including graft versus host, rejection. H52 is obtained from, orhas the identifying characteristics of, an antibody obtained from thecell line having ATCC accession number BB X. This cell line was placedon deposit for 30 years at the American Type Culture Collection (ATCC)in Rockville, Md. prior to Jun. 1, 1989.

[0015] Methods of Producing and Characterizing Monoclonal Antibodies

[0016] The general method used for production of hybridomas secretingmonoclonal antibodies is well known (Kohler, et al., European J. Imm.,6:292, 1976). Briefly, BALB/c mice were immunized with human splenicadherent cells and later boosted with the same type of cells. After 4days, the animals were sacrificed and the spleen cells fused with mousemyeloma P3X65 Ag8. Hybridomas were screened for antibody production andpositive clones were tested for reactivity towards human spleen tissuesections.

[0017] The present invention is directed to monoclonal antibodies, andhybridomas which produce them, which are reactive with the leukocyteadhesion receptor β-chain.

[0018] The isolation of hybridomas secreting monoclonal antibodies withthe reactivity of the monoclonal antibodies of the invention can beaccomplished using routine screening techniques to determine theelementary reaction pattern of the monoclonal antibody of interest.Thus, if a monoclonal antibody being tested reacts with the leukocyteadhesion receptor β-chain such that intercellular adhesion issuppressed, then the antibody being tested and the antibody produced bythe hybridomas of the invention are equivalent.

[0019] Alternatively, it is possible to evaluate, without undueexperimentation, a monoclonal antibody to determine whether it has thesame specificity as monoclonal antibody H52 of the invention bydetermining whether the monoclonal antibody being tested prevents H52from binding to a particular antigen, for example the LFA-1 receptorwith which H52 is normally reactive. If the monoclonal antibody beingtested competes with H52, as shown by a decrease in binding by H52, thenit is likely that the two monoclonal antibodies bind to the sameepitope.

[0020] Still another way to determine whether a monoclonal antibody hasthe specificity of H52 is to pre-incubate H52 with an antigen with whichit is normally reactive (for example, LFA-1 receptor), and determine ifthe monoclonal antibody being tested is inhibited in its ability to bindthe antigen. If the monoclonal antibody being tested is inhibited then,in all likelihood, it has the same epitopic specificity as themonoclonal antibody of the invention.

[0021] While the in vivo use of a monoclonal antibody from a foreigndonor species in a different host recipient species is usuallyuncomplicated, a potential problem which may arise is the appearance ofan adverse immunological response by the host to antigenic determinantspresent on the donor antibody. In some instances, this adverse responsecan be so severe as to curtail the in vivo use of the donor antibody inthe host. Further, the adverse host response may serve to hinder theintercellular adhesion-suppressing efficacy of the donor antibody. Oneway in which it is possible to circumvent the likelihood of an adverseimmune response occurring in the host is by using chimeric antibodies(Sun, et al., Hybridoma, 5 (Supplement 1):S17, 1986; Oi, et al., BioTechniques, 4(3): 214, 1986). Chimeric antibodies are antibodies inwhich the various domains of the antibodies' heavy and light chains arecoded for by DNA from more than one species. Typically, a chimericantibody will comprise the variable domains of the heavy (V_(H)) andlight (V_(L)) chains derived from the donor species producing theantibody of desired antigenic specificity, and the variable domains ofthe heavy (C_(H)) and light (C_(L)) chains derived from the hostrecipient species. It is believed that by reducing the exposure of thehost immune system to the antigenic determinants of the donor antibodydomains, especially those in the C_(H) region, the possibility of anadverse immunological response occurring in the recipient species willbe reduced. Thus, for example, it is possible to produce a chimericantibody for in vivo clinical use in humans which comprises mouse V_(H)and V_(L) domains coded for by DNA isolated from ATCC HB X, and C_(H)and C_(L) domains coded for with DNA isolated from a human leukocyte.

[0022] Under certain circumstances, monoclonal antibodies of one isotypemight be more preferable than those of another in terms of theirdiagnostic or therapeutic efficacy. For example, from studies onantibody-mediated cytolysis, it is known that unmodified mousemonoclonal antibodies of isotype gamma-2a and gamma-3 are generally moreeffective in lysing target cells than are antibodies of the gamma-1isotype. This differential efficacy is thought to be due to the abilityof the gamma2a and gamma-3 isotypes to more actively participate in thecytolytic destruction of target cells. Particular isotypes of amonoclonal antibody can be prepared either directly, by selecting fromthe initial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class-switch variants (Steplewski, etal., Proceedings of the National academy of Science, U.S.A., 82:8653,1985; Spira, et al., Journal of Immunological Methods, 74:307, 1984).Thus, the monoclonal antibodies of the invention would includeclass-switch variants having the specificity of monoclonal antibody H52which is produced by ATCC HB X.

[0023] When the monoclonal antibodies of the invention are used in theform of fragments, such as, for example, Fab and F(ab′)₂, and especiallywhen these fragments are therapeutically labeled, any isotype can beused since amelioration of the immune response disorders in thesesituations is not dependent upon complement-mediated cytolyticdestruction of those cells bearing the leukocyte adhesion receptor.

[0024] The term “immune response mediated disorder” denotes disorders inwhich the hosts' immune system contributes to the disease conditioneither directly or indirectly. Examples of disorders which are mediatedby the immune response includes AIDS, autoimmune disease, and graftrejection. As used herein, graft rejection encompasses both host versusgraft and graft versus host rejection.

[0025] The monoclonal antibodies of the invention can be used in anyanimal in which it is desirable to administer in vitro or in vivoimmunodiagnosis or immunotherapy. The term “animal” as used herein ismeant to include both humans as well as non-humans.

[0026] The term “antibody” as used in this invention is meant to includeintact molecules as well as fragments thereof, such as, for example, Faband F(ab′)₂, which are capable of binding the epitopic determinant.

DIAGNOSTIC USES

[0027] The monoclonal antibodies of the invention are suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. In addition, the monoclonal antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize monoclonal antibodies of theinvention are competitive and noncompetitive immunoassays in either adirect or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA) and the sandwich (immunometric) assay. Detectionof the antigens using the monoclonal antibodies of the invention can bedone utilizing immunoassays which are run in either the forward,reverse, or simultaneous modes, including immunohistochemical assays onphysiological samples.

[0028] The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of leukocyte adhesionfactor. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

[0029] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibody, or willbe able to ascertain such, using routine experimentation. Furthermore,the binding of these labels to the monoclonal antibody of the inventioncan be done using standard techniques common to those of ordinary skillin the art.

[0030] For purposes of the invention, the leukocyte adhesion factorβ-chain which is detected by the monoclonal antibodies of the inventionmay be present in biological fluids and tissues. Any sample containing adetectable amount of leukocyte adhesion factor β-chain can be used.Normally, a sample is a liquid such as urine, saliva, cerebrospinalfluid, blood, serum and the like, or a solid or semi-solid such astissues, feces, and the like.

[0031] Another technique which may also result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use such haptens as biotin, whichreacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, whichcan react with specific anti-hapten antibodies.

[0032] As used in this invention, the term “epitope” is meant to includeany determinant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

[0033] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the leukocyte adhesion receptor β-chain forwhich the monoclonal antibodies are specific.

[0034] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having leukocyte adhesion receptor is detectable compared to thebackground signal. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

[0035] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sexand extent of disease of the individual. The dosage of monoclonalantibody can vary from about 0.01 g/m² to about 20 mg/m², preferablyabout 0.1 mg/m² to about 10 mg/m².

[0036] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

[0037] For in vivo diagnosis radioisotopes may be bound to immunoglobineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.

[0038] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI.

[0039] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of an immune response mediated disorder in anindividual. Thus, by measuring the increase or decrease in the number ofleukocytes or changes in the concentration of antigen shed into variousbody fluids, it would be possible to determine whether a particulartherapeutic regimen aimed at ameliorating the immune response mediateddisorder is effective.

THERAPEUTIC USES

[0040] The term “ameliorate” denotes a lessening of the detrimentalaffect of the immune response mediated disorder in the animal receivingtherapy. The term “therapeutically effective” means that the amount ofmonoclonal antibody used is of sufficient quantity to ameliorate thecause of disease due to the immune response.

[0041] The monoclonal antibodies of the invention can also be used forimmunotherapy in an animal having an immune response mediated disordercaused by leukocytes which express leukocyte adhesion receptor β-chainwith epitopes reactive with the monoclonal antibodies of the invention.When used in this manner, the dosage of monoclonal antibody can varyfrom about 10 mg/m² to about 2000 mg/m².

[0042] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or labeled with a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener, et al., Science, 231:148, 1986) and can beselected to enable drug release from the monoclonal antibody molecule atthe target site. Examples of therapeutic agents which can be coupled tothe monoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

[0043] The drugs with which can be conjugated to the monoclonalantibodies of the invention include compounds which are classicallyreferred to as drugs such as for example, mitomycin C, daunorubicin, andvinblastine.

[0044] In using radioisotopically conjugated monoclonal antibodies ofthe invention for immunotherapy certain isotypes may be more preferablethan others depending on such factors as leukocyte distribution as wellas isotype stability and emission. If desired, the leukocytedistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the immune response mediated disorder someemitters may be preferable to other. In general, alpha and betaparticle-emitting radioisotopes are preferred in immunotherapy.Preferred are short range, high energy alpha emitters such as ²¹²Bi.Examples of radioisotopes which can be bound to the monoclonalantibodies of the invention for therapeutic purposes are ¹²⁵I, ¹³¹I,⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re.

[0045] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. However, ricin is a toxiclectin which has been used immunotherapeutically. This is accomplishedby binding the alpha-peptide chain of ricin, which is responsible fortoxicity, to the antibody molecule to enable site specific delivery ofthe toxic effect.

[0046] Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to aleukocyte expressing leukocyte adhesion factor β-chain for which themonoclonal antibodies of the invention are specific.

[0047] Other therapeutic agents which can be coupled to the monoclonalantibodies of the invention are known, or can be easily ascertained, bythose of ordinary skill in the art.

[0048] The dosage ranges for the administration of the monoclonalantibodies of the invention are those large enough to produce thedesired effect in which the symptoms of the immune response mediateddisorder are ameliorated. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary from about 0.01 mg/m² to about 2000 mg/m², preferably about 0.1mg/m² to about 500 mg/m²/dose, in one or more dose administrationsdaily, for one or several days.

[0049] Generally, when the monoclonal antibodies of the invention areadministered conjugated with therapeutic agents lower dosages, ascompared those used for in vivo immunodiagnostic imaging, can be used.

[0050] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally.

[0051] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

[0052] The invention also relates to a method for preparing a medicamentor pharmaceutical composition comprising the monoclonal antibodies ofthe invention, the medicament being used for therapy of immune responsemediated disorders due to leukocytes expressing leukocyte adhesionreceptor β-chain reactive with the monoclonal antibodies of theinvention.

[0053] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 PREPARATION OF ANTIADHESION MONOCLONAL FACTOR ANTIBODIES

[0054] Female Balb/c mice (6 to 8 weeks old) were injectedintraperitoneally with 1 human splenic adherent cells in phosphatebuffered saline. This treatment was repeated after 14 days and againafter 21 days. Four days after the final injection the spleen wasremoved from one of the immunized mice and a single cell suspensionprepared. The splenic cells were fused to Balb/c derived P3×653.Ag8myeloma cells with 50% polyethylene glycol after the method of Kohlerand Milstein (Nature:256:495, 1976). After establishing growinghybridoma colonies, supernatants from these cells were tested forantibodies against human antigens by immunohistochemistry on cryostatsections (4 μ) of frozen human spleens (Naiem, et al., J. Immun. Meth.,50:145, 1982). H52 (H52.G1.2) was selected for cloning and re-cloning byimmunohistochemistry, radioimmunoassays on human cells, and byradioimmunoprecipitation from human cells.

EXAMPLE 2 INHIBITION OF SYNCYTIUM FORMATION BY MONOCLONAL ANTIBODY

[0055] The effect of mAb on the fusion of 8E5 cells to PHA-blasts wasdetermined in a syncytium formation assay. The 8E5 and A3.01 cell lineswere maintained in complete medium (RPMI-1640 supplemented with 10% FBS(HyClone) and 10 mM HEPES). The 8E5 cell line is a surviving clone ofA.301 cells infected with LAV. 8E5 cells carry a single copy of theentire LAV genome but produce non-infectious virus particles due to apoint mutation in the reverse transcriptase gene. 8E5 cells express HIVenvelope glycoproteins and when mixed with CD4 -positive PHA-blasts andT cell lines produce cytopathic effects identical to those observed incultures of T cells infected with wild-type virus.

[0056] PHA-blasts were generated by incubating peripheral bloodmononuclear cells for 3 days in the presence of PHA (WellcomeDiagnostics) at a concentration of 0.25 μg/ml in complete medium. Cellswere washed 3 times with PBS and resuspended in complete medium at adensity of 5× 10⁶/ml. MAb were used in the form of purified IgG at aconcentration of 25 μg/ml. PHA-blasts were mixed with an equal volume(30 μl) of monoclonal antibody or medium in the wells of half-area96-well plates (Costar) and incubated for 30 minutes at 25° C. Thirty μlof 8E5 cells were then added followed by incubation for 10 hr at 37° C.in a humidified CO₂ incubator. Control wells consisted of PHA-blastsincubated with an equal number of uninfected A.301 cells. In the assay,syncytia or balloon cells consisting of 10 to 50 or more fused cellsform within 4 and 10 hours after mixing 8E5 cells with PHA blasts andCD4⁺ T cell lines, respectively. Continued incubation results in rapidsyncytia death as determined by vital dye exclusion.

[0057] To determine their effect on HIV-mediated cell fusion mAb againsthuman leukocyte antigens were added to co-cultures of PHA-blasts and 8E5cells. The mAb tested were: H52, anti-CD18 (LFA-1 β); MHM.24, anti-CD11a(LFA-1 α); H5A4, anti-CD11b (Mac-1 α); H5A5, anti-CD45 (leukocyte commonantigen); MHM.5, anti-HLA-A,B,C; Leu3a, anti-CD4. All antibodies areIgGl,k isotype.

[0058] As shown in FIG. 1, H52, against an epitope on the β-submit ofLFA-1 (CD 18), completely inhibited syncytium formation. Fusion was alsoblocked by a mAb (MHM.24) against the α-subunit of LFA-1 (CD11a).However, the mAb MHM.24 was less effective than mAb H52 since very smallsyncytia were rarely observed. H5A4, a mAb against a different member ofthe LAR family, Mac-1 (complement receptor type-3; CD11b), had no effecton the fusion of 8E5 cells to the PHA blasts. Also, fusion was notaffected by two mAb recognizing unrelated cell surface proteins: MHM.5,anti-HLA-A,B,C, and H5A5 , anti-leukocyte common antigen (CD45). Sincethese two antigens are expressed at equal or higher densities than LARon PHA blasts, the failure of the latter two antibodies to block fusionsuggests that inhibition by anti-LAR antibodies was not due to anon-specific steric effect. Leu3a, a mAb against CD4, which has beenpreviously shown to block binding of gp120 to CD4, completely inhibitedfusion of 8E5 to PHA-blasts (FIG. 1). Inhibition of fusion by Leu3a andthe absence of fusion between the PHA-blasts and uninfected A.301 cells(FIG. 1, control) confirmed that the fusion was mediated by HIV. Anumber of commercially available mAb against gp120 failed to inhibitfusion in the assay system. PHA-blasts and the 8E5 cells formed verylarge aggregates within 1 hr of mixing in the syncytium assay. Theseaggregates were completely inhibited by H52, MHM.24, and Leu3a, but notby the other mAb. The inhibition of syncytium formation by mAb H52 wasobserved whether the PHA blasts were mixed with 8E5 cells infected withthe mutant virus or the CEM T cell line infected with wild type HIV(HTLV-IIIB).

EXAMPLE 3 INHIBITION OF SYNCYTIUM FORMATION BY H52

[0059] PHA-blasts, generated as described in Example 1, were incubatedwith various concentrations of purified H52 or PLM-2 IgG before adding8E5 cells. PLM-2 is an IgGl,k mAb against CD18 which does not inhibitLFA-1 -mediated functions. The assay was carried out exactly asdescribed in Example 1. Syncytia were counted on an inverted microscopeusing a low power objective (40×) after adding trypan blue (0.1%). Datashown in FIG. 2 are the mean syncytia count/10 ⁶ 8E5 cells of duplicatewells.

[0060] The H52 mAb blocked 8E5 -PHA-blast fusion in a dose-dependentmanner with complete inhibition observed at concentrations above 3 μg/ml(FIG. 2). The inhibition of LFA-1 -mediated lymphocyte adhesionfunctions by mAb H52 shows a very similar dose-dependency. PLM-2, a mAbagainst a CD18 epitope not associated with LFA-1 adhesion functions, didnot affect fusion at any concentration (FIG. 2).

[0061] Studies were also done to determine the level at which fusion wasblocked by H52. PHA-blasts and 8E5 cells (2.5×10⁶) were incubated for 1hr on ice in 0.5 ml of complete medium alone or complete mediumcontaining purified H52 or PLM-2 IgG at 25 μg/ml. After pelleting thecells, unbound mAb was removed by washing 2 times with 10 ml of PBS. Theantibody-coated PHA blasts, and 8E5 cells were then resuspended incomplete medium and mixed with uncoated 8E5 cells and PHA blasts,respectively, followed by incubation at 37° C. for 10 hr as described inExample 1. Syncytia formation was scored as described above.

[0062] Previous studies have shown that inhibition of lymphocyteinteractions by anti-LFA-1 antibodies is a uni-directional effect evenwhen both cell types express LFA-1. To determine if the effect ofanti-LFA-1 mAb on syncytium formation was also uni-directional, LFA-1expression was analyzed by flow cytometry. Both 8E5 cells and PHA-blastsexpressed LFA-1, although the expression on 8E5 was substantially lessthan on the blasts.

[0063] Each cell type was pre-coated with mAb H52 or the control mAbPLM-2 and washed to remove unbound mAb before assaying syncytiumformation. Pre-coating PHA-blasts with H52 resulted in near completeinhibition of fusion while similar treatment of the 8E5 cells had noeffect (FIG. 3). Fusion was not affected by pre-coating either thePHA-blasts or the 8E5 cells with the control mAb. This result showedthat the anti-LFA-1 antibody blocked fusion at the level of thePHA-blast and not the HIV-infected 8E5 cells. This suggests that LAR onthe CD4⁺cells interacted with a ligand expressed on 8E5 cells.

EXAMPLE 4 INHIBITION OF HIV gp120 BINDING BY H52

[0064] Non-specific agents such as dextran sulfate that block theinteraction of HIV envelope glycoprotein gp120 with CD4 by stericeffects are known to inhibit HIV-mediated cell-cell fusion.Consequently, the binding of mAb H52 to LFA-1 on the surface ofCD4⁺-cells was tested to determine whether H52 interfered with thebinding of gp120 to CD4.

[0065] To purify gp120, HIV was pelleted (110,000×g, 1.5 hr) fromculture supernatants of infected PHA-blasts cells and washed once withPBS. The virus was resuspended in PBS and vortexed vigorously to shearoff gp120, followed by centrifugation at 110,000×g. The resultingsupernatant was concentrated using a 30,000 dalton cut-off Centriconfilter. The retained proteins, which consisted primarily of gp120 andbovine serum albumin (BSA; 10 to 30%), were radioiodinated using thestandard chloramine-T method. The labeled proteins (2 to 5 μCi/g) werediluted in PBS containing a high concentration of BSA (2%) to eliminatebinding of ¹²⁵I-BSA. CD4⁺-CEM cells (5×10⁵) were preincubated withLeu3a, H52, and PLM-2 mAb at 25 g/ml in complete medium (see Example 1)before adding 50 ng of radioiodinated gp120. Following a 1 hr incubationat 0° C. the cells were washed twice and bound radiolabel measured.Background binding was determined by preincubating cells with a 200-foldexcess of unlabeled gp120. Consistent with previous findings, cellspre-coated with the Leu3a mAb (anti-CD4) did not bind gp120 (FIG. 4). Incontrast, pre-coating cells with either mAb H52 or the control mAb PLM-2had no inhibitory effect on the binding of gp120. This resultdemonstrated that inhibition of syncytium formation by mAb H52 was notdue to interference with HIV receptor function since binding of gp120 toCD4 was not blocked by this mAb.

[0066] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made without departing from the spirit or scope of the invention.

1. A continuous hybridoma cell line capable of secreting monoclonalantibodies reactive with leukocyte adhesion receptor β-chain, whereinthe monoclonal antibodies suppress intercellular leukocyte adhesion. 2.The hybridoma of claim 1 , wherein the receptor is selected from thegroup consisting of LFA-1, Mac-1, and Leu M5.
 3. The hybridoma of claim1 , wherein the hybridoma is ATCC HB X and its isotype switch variants.4. A monoclonal antibody reactive with leukocyte adhesion receptor,wherein the β-chain monoclonal antibody inhibits intercellular leukocyteadhesion.
 5. The monoclonal antibody of claim 4 , wherein receptor isselected from the group consisting of LFA-1, Mac-1 and Leu M5.
 6. Themonoclonal antibody of claim 4 , having the specificity of a monoclonalantibody produced by hybridoma cell line ATCC HB X.
 7. The monoclonalantibody of claim 4 , wherein the monoclonal antibody is produced byhybridoma cell line ATCC HB X.
 8. A method of ameliorating an immuneresponse mediated disorder in an animal which comprises: administeringto the animal a therapeutically effective amount of an antibody, capableof suppressing intercellular leukocyte adhesion, wherein the antibodybinds to an epitope on the leukocyte adhesion receptor β-chain.
 9. Themethod of claim 8 , wherein the receptor is selected from the groupconsisting of LFA-1, Mac-1, and Leu M5.
 10. The method of claim 8 ,wherein the disorder is selected from the group consisting of AIDS,antoimmune disease, and graft rejection.
 11. The method of claim 8 ,wherein the monoclonal antibody has the specificity of the monoclonalantibody produced by ATCC HB X.
 12. The method of claim 8 , wherein theantibody is produced by hybridoma cell line ATCC HB X.
 13. The method ofclaim 8 , wherein the administration is parenteral.
 14. The method ofclaim 13 , wherein the parenteral administration is by subcutaneous,intramuscular, intraperitoneal, intracavity, transdermal, or intravenousinjection.
 15. The method of claim 8 , wherein said administration is ata dosage of about 0.01 mg/kg/dose to about 2000 mg/kg/dose.
 16. Themethod of claim 8 , wherein the monoclonal antibody is therapeuticallylabelled.
 17. The method of claim 16 , wherein the therapeutic label isselected from the group consisting of a radioisotope, a drug, a lectin,and a toxin.
 18. A method of detecting leukocyte adhesion receptor whichcomprises contacting a source suspected of containing the factor with adiagnostically effective amount of detectably labeled monoclonalantibody, or fragment thereof, having the specificity of monoclonalantibody H52 and its isotype switch variants and determining whether theantibody binds to the source.
 19. The method of claim 18 , wherein theantibody is produced by hybridoma cell line ATCC HB X.
 20. The method ofclaim 18 , wherein the detecting is in vivo.
 21. The method of claim 20, wherein the detectable label is selected from the group consisting ofa radioisotope and a paramagnetic label.
 22. The method of claim 18 ,wherein the detecting is in vitro.
 23. The method of claim 22 , whereinthe detectable label is selected from the group consisting of aradioisotope, a fluorescent compound, a colloidal metal, achemiluminescent compound, a bioluminescent compound and an enzyme.